Biol Reprod Lalor Postdoctoral Fellowships -- Application Deadline January 15, 2009
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.
Agricola
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.
Biology of Reproduction 62, 1160-1167 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Articles

Identification of Components of the Intrafollicular Bradykinin-Producing System in the Porcine Ovary1

Takahiro Kiharaa, Atsushi Kimuraa, Akihiko Moriyamab, Iwao Ohkuboc, and Takayuki Takahashi2,a

a Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan b Division of Biomolecular Science, Institute of Natural Sciences, Nagoya City University, Nagoya, Japan c Department of Medical Biochemistry, Shiga University of Medical Science, Ohtsu, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
As a step in elucidating the biological role of plasma kallikrein (PK) present in the follicular fluid of mammalian ovaries, we examined pig ovary fluid to determine its constituent activators and substrates. Using the inactive precursor form of plasma kallikrein (prePK) as a substrate, we purified an enzyme capable of activating this protein. The prePK-activating enzyme was shown to be the active enzyme blood coagulation factor XIIa. We also isolated high molecular weight kininogen (HMW-K) from the same fluid. Incubation of HMW-K with the ovarian follicular fluid PK resulted in the production of the nanopeptide bradykinin (BK). Expression of prePK, blood coagulation factor XII, and HMW-K was examined by Northern blot analysis using ovary and liver poly(A)+ RNA. All these transcripts were found in the liver, but none were found in the ovary. In addition, it was found that BK levels in the fluid derived from the small follicles were approximately 6 times higher than those from medium and large follicles. These results demonstrate the presence of a BK-producing system in the ovarian follicles and suggest the physiological importance of this peptide hormone in the early stages of follicular development and at ovulation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The germ cells in the ovaries grow to maturity in individual follicles and are eventually liberated under hormonal controls. The accumulation of fluid in the follicular space is a characteristic feature of mature ovarian follicles in most mammals. The follicular fluid contains a variety of proteins whose origin is generally thought to be either the plasma or the follicle itself [1]. The occurrence of several proteolytic enzymes in the fluid has been documented [2–6].

We previously reported that the follicular fluid of porcine ovaries contains the serine proteinase follipsin [7, 8]. Our subsequent studies revealed that plasma kallikrein (PK) is structurally and functionally homologous to porcine follipsin [9, 10]. These findings prompted us to further investigate the biological significance of the presence of intrafollicular PK using porcine ovaries. In the study reported here, we demonstrated that the follicular fluid contains factor XIIa as an activator of PK and high molecular weight kininogen (HMW-K) as a substrate of PK. The current study also showed a significant change in the intrafollicular concentration of bradykinin (BK) during follicle maturation. These results strongly suggest that the BK-generating system plays an important role in the development of ovarian follicles as well as at ovulation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials

Two synthetic substrates, butyloxycarbonyl(Boc)-Gln-Arg-Arg-4-methylcoumaryl-7-amide (MCA) for prePK (inactive precursor form of plasma kallikrein)-activating enzyme and benzoyl-Arg-MCA for papain-inhibiting activity of HMW-K, were purchased from the Peptide Institute (Osaka, Japan). DE-52 and CM-52 were obtained from Whatman (Clifton, NJ). Benzamidine-Sepharose 6B was purchased from Amersham Pharmacia Biotech Inc. (Piscataway, NJ). An SG-120 C-12 reversed-phase HPLC column was obtained from Shiseido (Tokyo, Japan). Rabbit anti-human factor XII antiserum and rabbit anti-human HMW-K antiserum were purchased from Nordic Immunological Laboratories, Ltd. (Amsterdam, Netherlands) and The Binding Site, Ltd. (Birmingham, UK), respectively. Other reagents were obtained from the indicated sources.

Preparation of Porcine Follicular Fluid

Porcine ovaries were obtained from a local slaughterhouse within 30 min of the animals' deaths and were transferred to the laboratory on ice. Follicular contents were aspirated from porcine ovarian follicles with a syringe and a 26-gauge needle, and were centrifuged at 1000 x g for 20 min at 4°C to remove oocytes, granulosa cells, and debris. The resulting supernatant was further centrifuged at 10 000 x g for 20 min at 4°C to obtain a clear supernatant. In some experiments, small (1–3 mm in diameter), medium (4–6 mm), and large (6–8 mm) follicles of porcine ovaries were aspirated. Criteria for sizing the follicles were based on those of Akins and Morrissette [11].

Enzyme Assay

Enzyme activity of ovarian follicular fluid prePK-activating enzyme was assayed as previously described [10]. Briefly, aliquots of the fractions were incubated at 37°C in a volume of 32 µl in 0.1 M Tris-HCl (pH 8.0) with 5.6 pmol prePK for 5 min. The enzyme activities of the activated PK were then assayed with Boc-Gln-Arg-Arg-MCA. The release of fluorophore, 7-amino-4-methylcoumarin, was measured by spectrofluorometry using an excitation wavelength of 370 nm and an emission wavelength of 460 nm. Net activity was obtained by subtracting the appropriate control values. HMW-K was assayed for its inhibitory activity toward the cysteine protease papain [12] using benzoyl-Arg-MCA.

Purification of PrePK-Activating Enzyme

The follicular fluid of porcine ovaries (50 ml) was dialyzed against 50 mM sodium acetate (pH 6.0). After removing insoluble materials by centrifugation, the clear supernatant was applied to a CM-52 column (2.4 x 25 cm) previously equilibrated with 50 mM sodium acetate (pH 6.0). The column was washed extensively and eluted with a linear gradient of NaCl using 300 ml each of 50 mM sodium acetate (pH 6.0) and the same buffer containing 0.2 M NaCl. The active fractions (a total of 60 ml) were collected, pooled, dialyzed against 20 mM Tris-HCl (pH 7.4), and applied to a DE-52 column (0.8 x 3.8 cm) previously equilibrated with 20 mM Tris-HCl (pH 7.4). The retained proteins were eluted with a linear gradient of NaCl using 20 ml each of 20 mM Tris-HCl (pH 7.4) and the same buffer containing 0.4 M NaCl. The active fractions (a total of 11 ml) were collected, pooled, and applied directly to a benzamidine-Sepharose 6B column (0.8 x 1.9 cm) previously equilibrated with 20 mM Tris-HCl (pH 7.4) containing 0.2 M NaCl. The retained proteins were eluted with the buffer containing 50 mM benzamidine. The enzyme activity was assayed using Boc-Gln-Arg-Arg-MCA as substrate as described above. Fractions were monitored for protein at 300 nm in CM-52 column chromatography because the concentrations were too high to determine at 280 nm, while fractions from DE-52 and benzamidine-Sepharose 6B columns were monitored at 280 nm.

Purification of HMW-K

HMW-K was purified from the follicular fluid of porcine ovaries by a modification of the method described previously for the purification of plasma HMW-K [13]. Follicular fluid (50 ml) was diluted 6 times with 121 mM Tris-succinic acid buffer (pH 7.7) containing EDTA (1 mM), benzamidine (1 mM), {epsilon}-aminocaproic acid (1 mM), polybrene (50 µg/ml), and NaN3 (0.02%), and applied to a DE-52 column (2.4 x 20 cm). The column was eluted with 193 mM Tris-succinic acid buffer (pH 7.4) containing benzamidine (1 mM), {epsilon}-aminocaproic acid (1 mM), polybrene (50 µg/mL), and NaN3 (0.02%). The fractions (a total of 50 ml) having papain-inhibiting activity (determined using benzoyl-Arg-MCA as substrate) were collected, pooled, and applied directly to a zinc-chelate Cellulofine column (1.8 x 8 cm; Seikagaku Corp., Tokyo, Japan) equilibrated with 25 mM Tris-HCl (pH 8.0) containing benzamidine (1 mM), {epsilon}-aminocaproic acid (1 mM), histidine (20 mM), NaCl (0.2 M), and NaN3 (0.01%). After unretained materials were removed by washing with the above buffer, the column was eluted with 25 mM Tris-HCl (pH 8.0) containing benzamidine (1 mM), {epsilon}-aminocaproic acid (1 mM), histidine (20 mM), NaCl (0.2 M), NaN3 (0.01%), and 5 mM EDTA. Fractions were monitored for protein at 300 nm in DE-52 column chromatography because the concentrations were too high to determine at 280 nm, while fractions from the zinc-chelate Cellulofine column were monitored at 280 nm.

Electrophoresis and Western Blot Analysis

SDS-PAGE was performed using a 10% acrylamide gel according to the method of Laemmli [14] and was followed by silver staining. Proteins separated by SDS-PAGE were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA) using Towbin's transfer buffer [15]. The blotted membranes were treated with Block Ace (Dainippon Seiyaku, Tokyo, Japan) at room temperature for 1 h. The membranes were incubated with primary antibodies at a 1:2000 dilution and subsequently with appropriate biotinylated, secondary antibodies. The membrane was then incubated with streptavidin conjugated with horseradish peroxidase, and immunoreactive signals on the membrane were detected by means of an ECL Western blot detection kit (Amersham Pharmacia Biotech).

Amino Acid Sequence Analysis

Polypeptides separated by SDS-PAGE were electroblotted onto a PVDF membrane and analyzed for NH2-terminal amino acid sequences in a PE Applied Biosystems (Foster City, CA) model 477A sequenator [16].

cDNA Cloning of PrePK-Activating Enzyme

Total RNA was obtained from porcine liver using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Poly(A)+ RNA was isolated by oligo(dT)-cellulose column chromatography. A cDNA library was constructed in {lambda}gt10 vector (Takara, Tokyo, Japan) using Gigapack III Gold packaging Extract (Stratagene, La Jolla, CA). The library was screened using a 1001-base pair (bp) digoxigenin-labeled reverse transcription (RT)-polymerase chain reaction (PCR) product, which had been prepared by amplifying with porcine liver mRNA using a sense primer of 5'-CCGGGGARCCCTGCCACTTC-3' corresponding to nucleotides 97–116 and an antisense primer of 5'-GCGGCGATGTAGGGGTGCGC-3' corresponding to nucleotides 1097–1078 of bovine factor XII [17]. From the 4.5 x 105 plaques screened, 4 positives were obtained and subjected to sequence analysis. The longest cDNA clone thus obtained had an insert of 2027 bp that still lacked several nucleotides at the 5'-end region of the coding sequence.

To isolate the 5'-end region of factor XII mRNA, a 5'-rapid amplification of mRNA ends (5'-RACE) [18] was performed using a 5'-Full RACE Core Set (Takara) according to procedures recommended by the manufacturer. Briefly, cDNA was synthesized by priming with 5'-end phosphorylated 14-mer nucleotides (5'-TCAGAGAAGACAGC-3' corresponding to nucleotides 1108–1095 of porcine factor XII) using poly(A)+ RNA isolated from the liver. The first PCR amplification was performed using a sense primer of 5'-CTCAAGGAGAGGTGCTACAG-3' (corresponding to nucleotides 637–656) and an antisense primer of 5'-CTTATGCTTCCTGGGGTCT-3' (corresponding to nucleotides 90–72). The second PCR amplification was performed with a sense primer of 5'-GCCACCTACTGGAACATGAC-3' (corresponding to nucleotides 733–752) and an antisense primer of 5'-GTGGAATCAAAAGAGCTGACTC-3' (corresponding to nucleotides 64–43). The final PCR product was cloned into the pBluescript II phagemid and sequenced.

Nucleotide Sequence Analysis

DNA sequencing was performed using either a PE Applied Biosystems 373A or 377A DNA sequencer.

Northern Blot Analysis

The Northern blot techniques used were basically as described by Sambrook et al. [19]. Five-microgram portions of poly(A)+ RNA isolated from whole porcine ovary and liver were electrophoresed in a 1.2% agarose gel and transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH). The probes used were the 1.3-kilobase (kb) SacI/AccI fragment of porcine plasma kallikrein cDNA (corresponding to nucleotides 1279–1868, DNA Data Bank of Japan [DDBJ]/European Molecular Biology Laboratory [EMBL]/GenBank Data Bank accession number AB022425) and the 1.0-kb RT-PCR product (corresponding to nucleotides 127–1163, DDBJ/EMBL/GenBank Data Bank accession number AB022426) of porcine factor XII. For the analysis of porcine HMW-K, a 0.55-kb RT-PCR product was prepared. To this end, the PCR was conducted using total porcine liver RNA with a sense primer of 5'-GTACAAGATGAAGAGCGGGA-3' corresponding to nucleotides 1310–1329 and an antisense primer of 5'-TTTGGGGGAGGTTGTTTCTG-3' corresponding to nucleotides 1849–1830 of bovine HMW-K [20]. The product was confirmed to be a fragment of porcine HMW-K DNA by nucleotide sequencing. The blotted membrane was hybridized with [{alpha}32P]dCTP random-labeled probes at 42°C in 50% formamide, 5-strength Denhardt's solution, 5-strength SSPE (single-strength SSPE is 150 mM NaCl, 10 mM NaH2PO4, 1 mM Na/EDTA), 1% SDS, and 100 µg/ml herring sperm DNA, and was washed twice in double-strength SSC (single-strength SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)/0.05% SDS at 50°C, and once each in single-strength SSC/0.1% SDS and double-strength SSC at the same temperature.

HPLC Analysis of BK

The HMW-K sample (0.6 nmol) purified from the follicular fluid of porcine ovaries as described above was incubated at 37°C with ovarian PK (27 pmol) in 200 µl of 0.1 M Tris-HCl (pH 8.0) for 5 min. Incubation was terminated by adding trifluoroacetic acid at a final concentration of 0.1%. The samples were passed through a 0.4-µm filter, and 175-µl aliquots were applied to a reversed-phase HPLC column equilibrated with 0.1% trifluoroacetic acid. The column was then eluted with a linear gradient (0–70%) of acetonitrile containing 0.1% trifluoroacetic acid, and absorbance was monitored at 220 nm. The peak was collected for amino acid sequence analysis.

Quantification of Follicular Fluid BK

Follicular fluids aspirated from small, medium, and large follicles were immediately mixed with 4 volumes of ice-cold ethanol, and the mixtures were centrifuged to recover the supernatants. The precipitates were again extracted with 80% ethanol. The supernatants thus obtained were combined and evaporated to dryness. After the pellets were dissolved in a small volume of distilled water, the solutions were adjusted to pH 2 using 0.1 N HCl. These solutions were washed twice with 3 volumes of diethyl ether, and the aqueous phases were dried. The resulting pellets were dissolved in distilled water for the determination of BK. The quantities of BK and (1–5)-BK (Arg-Pro-Pro-Gly-Phe) in the samples prepared above were estimated by ELISA using Markit-M BK and Markit-M (1–5)-BK kits (Dainippon Seiyaku, Tokyo, Japan), respectively. BK and (1–5)-BK in plasma were also determined by the same procedure.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Structural Characterization of Ovarian Follicular Fluid PrePK-Activating Enzyme

We confirmed our previous observation [10, 21] that the ovarian follicular fluid contains an enzyme capable of activating prePK. In order to determine its chemical makeup, we purified the enzyme by a series of column chromatographic steps on CM-52, DE-52, and benzamidine-Sepharose 6B columns (Fig. 1). The purified enzyme showed two bands of 89 kDa and 84 kDa under nonreducing conditions in SDS-PAGE. In contrast, SDS-PAGE of the enzyme under reducing conditions gave a clear band at a position of 39 kDa and broad bands at around 52 kDa (Fig. 2A). The NH2-terminal amino acid sequence analysis of the 39-kDa polypeptide produced a single sequence of Ile-Val-Gly-Gly-Leu-Val-Ala-Leu-Pro-Gly-Ala-His-Pro-Tyr-Ile-Ala-Ala-Leu (18 amino acid residues). The sequence was found to be the same as the NH2-terminal amino acid sequence of the catalytic domain of guinea pig factor XIIa [22] and was highly homologous to those of the domain of bovine [17], human [23, 24], and mouse factor XIIa (DDBJ accession number X99571). These results strongly suggest that the activating enzyme is factor XIIa. Consistent with this idea, the 89-, 84-, and 39-kDa polypeptides separated in SDS-PAGE were indeed recognized with anti-human factor XIIa antibody as examined by Western blot analysis (Fig. 2B). To further characterize the enzyme structurally, we conducted cDNA cloning experiments. Using a 1-kb porcine factor XII cDNA fragment corresponding to nucleotides 97–1097 of the bovine cDNA [17], we isolated 4 clones from the liver cDNA library. From analysis of these clones and the results of 5'-RACE experiments, we established the nucleotide sequence of porcine factor XII (the nucleotide sequence is available from the DDBJ/EMBL/GenBank Data Bank under accession No. AB022426). It encodes a predicted protein of 643 amino acids having a putative signal peptide of 14 amino acid residues. As a whole, the amino acid sequence has 80.9% identity with that of bovine factor XII. It also shows a high degree of homology with human (71.9% identity) [23, 24], guinea pig (70.5% identity) [22], and mouse factor XII (67.9% identity; cited from DDBJ data base accession No. X99571). The NH2-terminal 18-amino acid residue sequence of the 39-kDa polypeptide described above perfectly corresponds to the residues Ile372 to Leu389 in the deduced sequence.



View larger version (29K):
[in this window]
[in a new window]
  		FIG. 1. Chromatographic purification of prePK-activating enzyme from porcine ovarian follicular fluid. A) Chromatography on a CM-52 column. Flow rate, 43 ml/h; fraction size, 10 ml; monitoring absorbance, 300 nm. B) Chromatography on a DE-52 column. Flow rate, 40 ml/h; fraction size, 1 ml; monitoring absorbance, 280 nm. C) Chromatography on a benzamidine-Sepharose 6B column. Flow rate, 24 ml/h; fraction size, 1 ml; monitoring absorbance, 280 nm. A horizontal bar indicates the fractions pooled



View larger version (77K):
[in this window]
[in a new window]
  		FIG. 2. Electrophoretic analysis of prePK-activating enzyme. A) SDS-PAGE analysis. The enzyme (1 µg protein) was electrophoresed on a 10% polyacrylamide gel in the presence of SDS under reducing (left lane) and nonreducing (right lane) conditions, and the separated polypeptides were silver-stained. The relative molecular masses (kDa) of the polypeptides are shown. B) Western blot analysis. The blotted polypeptides were analyzed using anti-human factor XII antiserum. The 42-kDa band indicated by an arrow is presumably an impurity. ßME, ß-mercaptoethanol

The NH2-terminal amino acid sequences of the 89-kDa and 84-kDa polypeptides, which were separated in SDS-PAGE under nonreducing conditions, were also determined by Edman degradation. Both polypeptides contained two sets of sequences. The 89-kDa polypeptide produced Ile/Val-Met/Val-Ala/Gly-Ser/Gly-Leu/Glu-, while the 84-kDa polypeptide produced Ile/Trp-Val/Ala-Gly/Tyr-Gly-Leu-. The results indicate that both the 89-kDa and 84-kDa polypeptides commonly contain an NH2-terminal amino acid sequence of Ile-Val-Gly-Gly-Leu-, a sequence corresponding to the first 5 residues of the 39-kDa polypeptide. Additional sequences existing in the 89-kDa and 84-kDa polypeptides were presumed to be Val-Met-Ala-Ser-Glu- and Trp-Ala-Tyr-X-Leu-, respectively. On the basis of these results, it is reasonable to conclude that factor XIIa purified from the follicular fluid of porcine ovary is a two-chain enzyme. The enzyme exists in two different forms with a common COOH-terminal catalytic domain starting at Ile383. The two forms of the enzyme differ only in their NH2-terminal domain. The NH2 terminus of the polypeptides is either Val29 or Trp83 (Fig. 3). The 52-kDa and 46-kDa polypeptides separated in SDS-PAGE (Fig. 2A) are most likely the NH2-terminal domains of the two isoforms, although an NH2-terminal amino acid sequence analysis of the polypeptides was not conducted. The reason why these polypeptides were not visible in Western blot analysis (Fig. 2B) is probably that the anti-human factor XIIa antibody used in this experiment poorly recognizes the NH2-terminal domain of porcine factor XIIa.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 3. Schematic representation of the two isoforms of factor XIIa. Both forms of porcine ovary factor XIIa consist of a two-chain enzyme having a common COOH-terminal catalytic domain of 39 kDa. The location of the active site Ser is indicated in the 39-kDa polypeptide

HMW-K from Porcine Ovarian Follicular Fluid

After identifying PK and factor XIIa in the follicular fluid, we attempted to determine whether HMW-K, a physiological substrate of PK, coexists in the same fluid. A preliminary Western blot experiment using anti-human HMW-K antibody strongly suggested that the fluid indeed contained proteins cross-reactive with the antibody. Therefore, we attempted to purify the proteins from the follicular fluid of porcine ovaries by a combination of column chromatographies on DE-52 and zinc-chelate Cellulofine (Fig. 4). The results of the SDS-PAGE analysis of the purified protein are shown in Figure 5. The 140-kDa and 105-kDa polypeptides were separated on SDS-PAGE under nonreducing conditions. Similar results were reported for HMW-K purified from porcine plasma, except that the apparent molecular mass of the larger polypeptide was 200 kDa [13]. These polypeptides (140 kDa and 105 kDa) are presumed to be dimeric and monomeric forms of HMW-K, respectively [25]. As shown in Figure 5B, anti-human HMW-K antibody recognized the 120-kDa polypeptide (reducing) as well as the 140-kDa and 105-kDa polypeptides (nonreducing). The 60-kDa polypeptide stained in SDS-PAGE under reducing conditions was judged to be an impurity, since the polypeptide was not recognized by the antibody. To further identify the purified protein as HMW-K, its reactivity to PK was examined. Incubation with PK produced a single HPLC peak at an elution time of 16 min (Fig. 6). The amino acid sequence of the peak was found to be Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, which is the same as that of human BK [26]. The electrophoretic data indicated that the purified sample contained virtually no nicked or kinin-free HMW-K.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 4. Chromatographic purification of HMW-K from porcine ovarian follicular fluid. A) Chromatography on a DE-52 column. Flow-through fractions are not shown. Flow rate, 62 ml/h; fraction size, 10 ml; monitoring absorbance, 300 nm. B) Chromatography on a zinc-chelate Cellulofine column. Flow rate, 32 ml/h; fraction size, 2 ml; monitoring absorbance, 280 nm. Elution was started at the position indicated by an arrow, and the fractions were assayed for protein and papain-inhibiting activity. A horizontal bar indicates the fractions pooled



View larger version (49K):
[in this window]
[in a new window]
  		FIG. 5. Electrophoretic analysis of ovarian follicular fluid HMW-K. A) SDS-PAGE analysis. The HMW-K sample purified as in Figure 4 was electrophoresed on a 10% polyacrylamide gel in the presence of SDS under reducing (left lane) and nonreducing (right lane) conditions. One microgram of protein was applied. The separated polypeptides were silver-stained. Relative molecular masses (kDa) of the polypeptides are shown. B) Western blot analysis. The blotted polypeptides were analyzed using anti-human HMW-K antiserum



View larger version (30K):
[in this window]
[in a new window]
  		FIG. 6. BK liberation from the follicular fluid HMW-K by porcine ovarian PK. The HMW-K sample incubated with porcine PK was analyzed on a reversed-phase HPLC column in a 0.1% trifluoroacetic acid/acetonitrile system. The peak eluted at 16 min (indicated by an arrow) was collected and analyzed for its amino acid sequence. A) HMW-K was incubated alone; B) PK was incubated alone; C) HMW-K was incubated with PK

It must be noted that the flow-through fractions of DE-52 column chromatography (Fig. 4A) exhibited a significant papain-inhibiting activity (data not shown), indicating the presence of another type of kininogen in the follicular fluid. However, this fraction was found not to be retained on the zinc-chelate Cellulofine column. These results strongly suggest that the fluid also contains low-molecular weight kininogen (LMW-K).

mRNA Detection of the Components of the BK-Producing System

The above results clearly indicate that the BK-producing system is present in the follicles of porcine ovary. We next examined whether the components are expressed in the ovary. A Northern blot analysis was performed with 32P-labeled respective porcine cDNA fragments using total RNA of the porcine ovary and liver. Transcripts of 2.40 kb (PK), 2.05 kb (factor XII), and 3.05 kb (HMW-K) were not found in the ovary, whereas a significant expression of the transcripts was observed with the liver total RNA (Fig. 7).



View larger version (37K):
[in this window]
[in a new window]
  		FIG. 7. Northern blot analysis of mRNA isolated from porcine ovary and liver. Poly(A)+ RNAs were prepared from pig ovary and liver. A 5-µg portion of poly(A)+ RNAs from the tissues was analyzed for mRNAs of PK (A), factor XII (B), and HMW-K (C) by hybridization with the respective porcine cDNA fragments (see Materials and Methods)

BK Concentration in the Fluid

The amounts of BK and of the degraded product (1–5)-BK were determined using follicular fluids derived from various sizes of porcine ovarian follicles. As shown in Figure 8, BK levels were 6 times higher in the fluid derived from small follicles than in fluids from medium and large follicles. The plasma levels of this peptide were comparable to its intrafollicular levels in the medium- and large-sized follicles. Similarly, the (1–5)-BK levels were the greatest in the small follicles; these levels drastically decreased as the follicles grew. This degradation product was present in the plasma at a significantly higher level than in the fluids of the medium- and large-sized follicles.



View larger version (31K):
[in this window]
[in a new window]
  		FIG. 8. Quantification of BK and (1–5)-BK in porcine follicular fluid. Follicular fluid was prepared from small (S), medium (M), and large follicles (L) of porcine ovaries, and the plasma (P) was obtained from the blood of a female pig. The samples were analyzed for quantities of BK and (1–5)-BK by ELISA. A) Determination of BK. The values are means ± SEM of three determinations. *Significantly different (P < 0.01) from medium and large follicles, and from plasma. B) Determination of (1–5)-BK. The values are means ± SEM of three determinations. *Significantly different (P < 0.01) from medium and large follicles, and significantly different (P < 0.05) from plasma. **Significantly different (P < 0.02) from medium and large follicles


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous studies have demonstrated an increase in ovarian kinin-producing activity during ovulation [27–29]. Moreover, it has been established that BK induces ovulation in perfused rabbit ovaries [30–32] and potentiates the action of LH [33]. Despite the fact that the role of BK in mammalian ovaries is well recognized, little is known about how the peptide hormone is produced in vivo. As a first step in approaching this problem, we investigated whether the components responsible for the production of BK are present in individual ovarian follicles. The current results indicate that the follicular fluid of porcine ovary contains factor XIIa as an activator of the inactive precursor PK as well as HMW-K as a substrate of PK. To the best of our knowledge, this is the first biochemical demonstration of the presence of an intrafollicular BK-producing system in the ovary. Our present finding that not only the components for a BK-producing system, but also the product BK, exist in the follicular fluid of the porcine ovary strongly suggests that BK can be produced within ovarian follicles. However, its production in the follicles still remains to be demonstrated.

In this study, the presence of LMW-K in the follicular fluid of the porcine ovary was implied. This points to the possibility that intrafollicular kallidin (lysyl-BK) might also be produced from LMW-K. A tissue kallikrein is generally thought to catalyze the production of kinin from LMW-K. Several investigators have indeed reported an increase in tissue kallikrein activity [27–29, 34] with a concomitant elevation of LMW-K [28] in rat ovary extract during ovulation. At present, however, it is not known to what extent the LMW-K/tissue kallikrein system contributes to kinin formation within porcine ovarian follicles.

Upon Northern blot analysis of the total RNA fraction prepared from porcine ovary, no clear transcript was detected for any components of the BK-producing system. On the other hand, the total RNA fraction from the liver gave detectable signals for all of the components.

These observations, together with the generally accepted idea that prePK, HMW-K, and factor XII in the plasma are synthesized in and secreted from the liver cells, might lead to the conclusion that the components constituting the intrafollicular BK-producing system are of hepatic origin. If this were the case, an unknown mechanism would be responsible for moving such compounds into the follicles from the circulatory system surrounding them. Alternatively, it may be that these protein components for the intrafollicular BK-producing system are synthesized within the follicle itself, but that Northern blot analysis is not sensitive enough to detect the respective transcripts. Since poly(A)+ RNAs were extracted from a whole ovary, transcripts of the components, which may be expressed specifically in follicle cells in low abundance, may have been diluted out during the analysis. At any rate, the derivation of the intrafollicular components of the system is not clear at present. Clearly, further investigations will be needed to determine the site of synthesis.

From the results shown in Figure 8A, the average intrafollicular BK concentrations were calculated to be approximately 1.4 x 10-11, 2.8 x 10-12, and 2.1 x 10-12 M for small, medium, and large follicles, respectively, assuming that the molecular weight of BK is 1060. In this context, it is interesting to note that the reported Kd values of BK receptors for the ligand are 10-10–~10-8 M [35]. These considerations suggest that the BK concentration in the fluid, particularly in small growing follicles, could be within a physiological range. Follicular growth in the mammalian ovary is accompanied by a striking accumulation of fluid within the follicles that mainly originates from plasma. Because BK causes both a dilation of blood vessels and increases in vascular permeability, one possible explanation for the involvement of the peptide hormone is that its intrafollicular production would result in an increased permeability of the capillaries of small growing follicles, which in turn would enhance the transfer of plasma substances into the surrounding extravascular spaces and also into the follicular antrum. It should be noted, however, that enlargement of follicles due to increased permeability is observed throughout follicular development and is particularly pronounced in the later stages. The present finding that follicular BK levels in the follicles decreased with the growth of follicles would appear to be inconsistent with the above hypothesis, indicating the presence of a more complex mechanism(s) for follicular growth. Our results also suggest that small follicles are able to sequester BK. BK is known to be degraded rapidly in vivo, with a half-life of about 16 sec [36]. Although enzymes responsible for its degradation within the follicles are not known at present, the elevated BK level in small follicles may be due to increased activities of the BK-generating enzymes and/or decreased activities of putative BK-degrading enzymes, as compared with those in medium and large follicles.

Our present finding that BK is concentrated more in the fluid of small follicles than in the fluid of larger follicles also tempts us to speculate that this peptide hormone may play a role in the atresia of follicles, since small follicles in the antral stages are more often destined for programmed cell death by atresia than are large preovulatory follicles [37, 38]. Therefore, it would be very interesting to examine whether BK is involved in the selection and atresia of ovarian follicles.

Hellberg et al. [32] observed that BK potentiates the ovulatory response to LH in in vitro-perfused rat ovaries. In addition, BK treatment resulted in increased ovarian tissue levels of prostaglandin E2 and prostacyclin. On the basis of these results, they proposed that vasodilatory mediators, such as prostacyclin, may potentiate vascular leakage without macromolecular permeability. To examine whether this is indeed an in vivo mechanism, it will be necessary to determine the BK concentration in graafian follicles that are about to ovulate, for which the highest BK levels are expected. The ovarian follicles used in this study were exclusively in the preovulatory stages of follicular development, and no ovulating follicles were available. Therefore, an in vitro experiment using the whole isolated follicles treated with LH will be needed to confirm the above hypothesis.

In conclusion, this study demonstrated the presence of components of the BK-producing system as well as the product BK in the ovarian follicles, suggesting important roles of BK not only in the early stages of follicular development but also at ovulation.


   ACKNOWLEDGMENTS
 
We wish to thank Dr. Norio Suzuki (Division of Biological Science) for his valuable suggestions.


   FOOTNOTES
 
First decision: 29 September 1999.

1 This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. T.K. and A.K. are supported by Research Fellowships of the Japan Society for the Promotion of Science for Young Scientists. Back

2 Correspondence. FAX: 81 11 706 4851; ttakaha{at}sci.hokudai.ac.jp Back

Accepted: December 9, 1999.

Received: August 24, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. McNatty KP. Follicular fluid. In: Jones RE (ed.), The Vertebrate Ovary. New York: Plenum Press; 1978: 215–259.
  2. Tsafriri A, Denkel N. Molecular mechanisms in ovulation. In: Findlay JK (ed.), Molecular Biology of The Female Reproductive System. San Diego: Academic Press; 1994: 207–258.
  3. Espey LL, Coons PJ. Factors which influence ovulatory degradation of rabbit ovarian follicles. Biol Reprod 1976; 14:232–245.
  4. Beers WH. Follicular plasminogen and plasminogen activator and the effect of plasmin on ovarian follicle wall. Cell 1975; 6:379–386.[CrossRef][Medline]
  5. Beers WH, Strickland S, Reich E. Ovarian plasminogen activator: Relationship to ovulation and hormonal regulation. Cell 1975; 6:387–394.[CrossRef][Medline]
  6. Reich E, Miskin R, Tsafriri A. Follicular plasminogen activator: involvement in ovulation. Endocrinology 1985; 116:516–521.[Abstract/Free Full Text]
  7. Hamabata T, Okimura H, Yokoyama N, Takahashi K, Takahashi T. Purification, characterization, and localization of follipsin, a novel serine proteinase from the fluid of porcine ovarian follicles. J Biol Chem 1994; 269:17899–17904.[Abstract/Free Full Text]
  8. Ohnishi J, Kihara T, Hamabata T, Takahashi K, Takahashi T. Cleavage specificity of porcine follipsin. J Biol Chem 1995; 270:19391–19394.[Abstract/Free Full Text]
  9. Ohnishi J, Murata M, Kohyama K, Yoshida H, Wada S, Makinoda S, Fujimoto S, Takahashi T. Follicular fluid from human ovaries contains plasma kallikrein in free form and in complex with {alpha}2-macroglobulin. Biomed Res 1997; 18:161–170.
  10. Kihara T, Ohnishi J, Kohyama K, Moriyama A, Takahashi T. Identification and activation of profollipsin, a latent precursor form of porcine follipsin. Eur J Biochem 1997; 245:392–397.[Medline]
  11. Akins EL, Morrissette MC. Gross ovarian changes during estrous cycle of swine. Am J Vet Res 1968; 29:1953–1957.[Medline]
  12. Sasaki M, Taniguchi K, Minakata K. Multimolecular forms of thiol proteinase inhibitor in human plasma. J Biochem 1981; 89:169–177.[Abstract/Free Full Text]
  13. Tani Y, Ohkubo I, Higashiyama S, Kunimatsu M, Sasaki M. Porcine high molecular weight kininogen: its purification and properties as thiol proteinase inhibitor as compared to human high molecular weight kininogen. Comp Biochem Physiol 1987; 88B:429–441.
  14. Laemmili UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227:680–685.[CrossRef][Medline]
  15. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 1979; 76:4350–4354.[Abstract/Free Full Text]
  16. Matsudaira P. Sequence from picomole quantities of proteins electro-blotted onto polyvinylidene difluoride membranes. J Biol Chem 1987; 262:10035–10038.[Abstract/Free Full Text]
  17. Shibuya Y, Semba U, Okabe H, Kambara T, Yamamoto T. Primary structure of bovine Hageman factor (blood coagulation factor XII): comparison with human and guinea pig molecules. Biochim Biophys Acta 1994; 1206:63–70.[CrossRef][Medline]
  18. Frohman MA, Dush MK, Martin GR. Rapid production of full-length cDNA from rare transcripts: amplification using a single gene-specific oligonucleotide primer. Proc Natl Acad Sci USA 1988; 85:8998–9002.[Abstract/Free Full Text]
  19. Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning. Cold Spring: Cold Spring Harbor Laboratory Press; 1989.
  20. Kitamura N, Takagaki Y, Furuto S, Tanaka T, Nawa H, Nakahishi S. A single gene for bovine high molecular weight and low molecular weight kininogens. Nature 1983; 305:545–549.[CrossRef][Medline]
  21. Kihara T, Kimura A, Ohnishi J, Takahashi T. A latent precursor form of follipsin and its activating enzyme. In: Kawashima S, Kikuyama S (eds.), Advances in Comparative Endocrinology, vol. 2. Bologna: Monduzzi Editore; 1997: 1545–1548.
  22. Semba U, Yamamoto T, Kunisada T, Shibuya Y, Tanase S, Kambara T, Okabe H. Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule. Biochim Biophys Acta 1992; 1159:113–121.[CrossRef][Medline]
  23. Cool DE, Edgell C-JS, Louie GV, Zoller MJ, Brayer GD, MacGillivray RTA. Characterization of human blood coagulation factor XII cDNA. Prediction of the primary structure of factor XII and the tertiary structure of beta-factor XIIa. J Biol Chem 1985; 260:13666–13676.[Abstract/Free Full Text]
  24. Que BG, Davie EW. Characterization of a cDNA coding for human factor XII (Hageman factor). Biochemistry 1986; 25:1525–1528.[CrossRef][Medline]
  25. Higashiyama S, Ohkubo I, Ishiguro H, Kunimatsu M, Sawaki K, Sasaki M. Human high molecular weight kininogen as a thiol proteinase inhibitor: presence of the entire inhibition capacity in the native form of heavy chain. Biochemistry 1986; 25:1669–1675.[CrossRef][Medline]
  26. Regoli D, Barabé J. Pharmacology of bradykinin and related kinins. Pharmacol Rev 1980; 32:1–46.[Medline]
  27. Gao X, Greenbaum LM, Mahesh VB, Brann DW. Characterization of the kinin system in the ovary during ovulation in the rat. Biol Reprod 1992; 47:945–951.[Abstract]
  28. Smith C, Perks AM. The kinin system and ovulation: changes in plasma kininogen, and in kinin-forming enzymes in the ovaries and blood of rats with 4-day estrous cycles. Can J Physiol Pharmacol 1983; 61:736–742.[Medline]
  29. Espey LL, Miller DH, Margolius HS. Ovarian increase in kinin-generating capacity in PMSG/hCG-primed immature rat. Am J Physiol 1986; 251:E362-E365.
  30. Lambertsen CJ Jr, Greenbaum DF, Wright KH, Wallach EE. In vitro studies of ovulation in the perfused rabbit ovary. Fertil Steril 1976; 27:178–187.[Medline]
  31. Yoshimura Y, Espey LL, Hosoi Y, Adashi T, Atlas SJ, Ghodgaonkar RB, Dubin NH, Wallach EE. The effects of bradykinin on ovulation and prostaglandin production by the perfused rabbit ovary. Endocrinology 1988; 122:2540–2546.[Abstract/Free Full Text]
  32. Hellberg P, Larson L, Olofsson J, Hedin L, Brännström M. Stimulatory effects of bradykinin on the ovulatory process in the in vitro-perfused rat ovary. Biol Reprod 1991; 44:269–274.[Abstract]
  33. Brännström M, Hellberg P. Bradykinin potentiates LH-induced follicular rupture in the rat ovary perfused in vitro. Hum Reprod 1989; 4:475–481.[Abstract/Free Full Text]
  34. Espey LL, Tanaka N, Winn V, Okamura HJ. Increase in ovarian kallikrein activity during ovulation in the gonadotrophin-primed immature rat. J Reprod Fertil 1989; 87:503–508.[Abstract/Free Full Text]
  35. Hall JM. Bradykinin receptor. Gen Pharmacol 1997; 28:1–6.[Medline]
  36. Balldin G, Gustafsson EL, Ohlsson K. Influence of plasma protease inhibitors and trasylol on trypsin-induced bradykinin-release in vitro and in vivo. Eur Surg Res 1980; 12:260–269.[Medline]
  37. Erickson GF, Magoffin DA, Dyer CA, Hofeditz C. The ovarian androgen producing cells: a review of structure/function relationships. Endocr Rev 1985; 6:371–399.[Abstract/Free Full Text]
  38. Byskw AG. Follicular atresia. In: Jones RE (ed.), The Vertebrate Ovary. New York: Plenum Press; 1978: 533–562.



This article has been cited by other articles:


Home page
 ReproductionHome page
P. Singh, A. Krishna, and R. Sridaran
Localization of gonadotrophin-releasing hormone I, bradykinin and their receptors in the ovaries of non-mammalian vertebrates
Reproduction, May 1, 2007; 133(5): 969 - 981.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
K.A. Vonnahme, S.C. Fernando, J.W. Ross, M.D. Ashworth, U. DeSilva, J.R. Malayer, and R.D. Geisert
Porcine Endometrial Expression of Kininogen, Factor XII, and Plasma Kallikrein in Cyclic and Pregnant Gilts
Biol Reprod, January 1, 2004; 70(1): 132 - 138.
[Abstract] [Full Text] [PDF]

Home page
 Biol. Reprod.Home page
A. Kimura, T. Kihara, R. Ohkura, K. Ogiwara, and T. Takahashi
Localization of Bradykinin B2 Receptor in the Follicles of Porcine Ovary and Increased Expression of Matrix Metalloproteinase-3 and -20 in Cultured Granulosa Cells by Bradykinin Treatment
Biol Reprod, November 1, 2001; 65(5): 1462 - 1470.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow My Folders
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.
Agricola
Right arrow Articles by Kihara, T.
Right arrow Articles by Takahashi, T.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS